Process For Preparing Liquid Mixtures Of Known pH And Salt Concentration

ABSTRACT

A method of preparing a liquid mixture for use in a liquid chromatography system is provided. The mixture comprises one or more acids, one or more bases, one or more salts, and one or more solvents, and the method comprises the steps of: i) calculating pH and/or salt concentration at a particular time t from a user-determined gradient function; and ii) based on the values obtained in step (i), calculating percent acid, percent base, percent salt and percent solvent in the liquid mixture at time t. A liquid chromatography system incorporating such method is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/348,280, filed on May 26, 2010, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to liquid mixtures. More specifically, theinvention pertains to methods of preparing liquid mixtures of known pHand salt concentration for use in various methods in analyticalchemistry, such as liquid chromatography systems.

BACKGROUND OF THE INVENTION

Liquid chromatography is a form of chromatography used frequently inbiochemistry and analytical chemistry to separate, identify, andquantify compounds based on their properties such as polarity and theirinteractions with a stationary phase. Liquid chromatography can beperformed using planar or column techniques. In both cases, the systemincludes a chromatographic device with a stationary phase, a means formoving a mobile phase (solvent carrying compounds of interest) throughthe device (such as a pump, or gravity), and a detector that creates anelectrical signal that identifies a particular compound based on thetime of the signal and the amount of the compound based on the intensityof the signal. The detector may also provide other characteristicinformation (e.g. UV/Vis spectroscopic data for a compound if soequipped). Retention time of a compound in the device varies dependingon the strength of the compound's interactions with the stationary andmobile phases, the ratio/composition of the mobile phase that is used,and the flow rate of the mobile phase.

The composition of the mobile phase flowing through the chromatographicdevice is critical to obtaining the required separation of compounds.For example, in ion exchange chromatography, the pH and/or saltconcentration of the mobile phase changes over the course of theseparation, to elute different compounds at different times. Prior artsystems allowed users to blend multiple solvents to create mobile phasesof particular pH and/or salt concentrations. However, in these systemsthe user was required to know not only the pH and salt concentrations oftheir particular solvents, but also the pH and salt concentrations thatwould result from mixing the solvents in various proportions. It wouldbe desirable to have a system and method for automatically calculatingand blending the solvents in the desired proportions, to produce aparticular pH and/or salt concentration specified by a user, and forvarying the proportions of solvents over the course of the elution.

SUMMARY OF THE INVENTION

Accordingly, in one aspect the invention provides a method of preparinga liquid mixture, the mixture comprising one or more acids, one or morebases, one or more salts, and one or more solvents, the methodcomprising the steps of: i) calculating pH and/or salt concentration ata particular time t from a user-determined gradient function; and ii)based on the values obtained in step (i), calculating percent acid,percent base, percent salt and percent solvent in the liquid mixture attime t.

In additional aspects, the invention also provides a computer programfor determining relative proportions in a liquid mixture according tothe above algorithm, wherein the proportions are used for controlling aliquid mixture preparation device. Also provided is an eluentpreparation device comprising i) a liquid mixture preparation devicecomprising a mixed liquid outlet port and a plurality of inlet portsconnected to component sources of at least one of an acid, a base, asalt, and a solvent, and ii) a mixer control unit arranged to controlthe relative component proportions supplied through the inlet ports ofthe liquid mixture preparation device, wherein the mixer control unit i)calculates pH and/or salt concentration at a particular time t from auser-determined gradient function; and ii) based on the values obtainedin step (i), calculates percent acid, percent base, percent salt andpercent solvent in the liquid mixture at time t.

A liquid chromatography system comprising an eluent preparation deviceis also provided.

DESCRIPTION OF THE DRAWINGS

The invention is further illustrated by the following drawings in which:

FIG. 1 is a diagram of different curves that are used to change the pHor salt concentration over the course of an elution.

FIG. 2 is a chromatogram showing separation according to the inventionas described in the example.

FIG. 3 is a chromatogram showing separation according to the inventionas described in the example.

DETAILED DESCRIPTION OF THE INVENTION

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about”, even if the term does notexpressly appear. Also, any numerical range recited herein is intendedto include all sub-ranges subsumed therein.

The chromatographic system uses four stock solvents to create thedesired pH-salt gradients for an elution, the solvents comprising anacid solution, a base solution, a salt solution and a solvent solutionthat is typically water, but may contain other additives. In someseparations an organic solvent is substituted for the salt. Thus, asused herein, including in the claims, the term “salt” is understood tomean a salt solution, or its substitute, an organic solvent solution.

These four stock solutions are metered in different relativeconcentrations to generate a given pH-salt concentration condition forthe delivered mobile phase at a given point of time.

In an embodiment of the invention, four different types of data areentered by the user into the computer program which controls operationof the chromatography system and specifically delivery of the correctsolutions at the pump: 1) a pH-salt gradient table; 2) an empirical pHcalibration table (or pK_(a)); 3) molar concentrations of acid, base andsalt stock solutions; and 4) the desired acid+base molar concentrationthat will be delivered to the stationary phase.

A pH look-up table is used by the software to calculate the percentageflow from acid and base lines during the elution. The look-up table iscreated by the software either from a user-input pK_(a) or from auser-input empirical calibration table. The empirical calibration tableis created by the user by mixing the 2-4 solutions in known proportionsand measuring the pH. The software then uses these input values tocalculate the percent acid and base (described further below) at a giventime point in the gradient table.

The gradient that is delivered to the stationary phase is determined inthe following manner, based on the user-entered information.

A pH-salt gradient table has values for 1) flow rate, 2) pH, 3) changein pH, (referred to herein as a “pH curve”), 4) salt concentration and5) change in salt concentration (referred to herein as the “saltcurve”), for each time t during the course of the separation. Typically,a user will prepare a table having 5 to 6 rows, for times t₀ to t₅ (ort₆). An example of a pH-salt gradient table is shown in Table 1:

TABLE 1 Time Flow pH Salt Salt (min) (mL/min) pH Curve Conc. Curve Ink.0.5 6.8 . . . 0.05 . . . 10 0.5 6.8 6 0.8 6 12 0.5 6.8 6 0.8 6 15 0.56.8 11 0.05 11

Each time period, for example the time from t₁ to t₂, is referred to a“time segment”. The pH or salt curve determines how the pH or saltconcentration will change over that particular time segment. Examples ofcurves, which can be used to vary both the pH and salt concentration,are depicted in the FIG. 1. The change can be linear, where halfwaythrough the time segment the percentages are halfway between thestarting and the ending percentages. The change may also occur morequickly at the beginning of the time segment and more slowly at the endof the segment, a convex curve. The opposite pattern, a concave curve isalso possible. The change can also be a step at either the beginning orthe end of the segment. The change of pH and salt concentration can beindependently varied or held constant from one time segment to another,depending on the needs of the user.

The pH-salt gradient table is prepared by the user based onexperimentation and prior experience with particular solvents andanalytes of interest, and is within the ability of one skilled in theart of chromatography systems.

The user enters the pK_(a) from which the look-up table is calculated,or they may enter an empirical calibration from which the pH look-upsare calculated, into the computer program. Optionally, the system caninclude pre-generated look-up tables for common buffer systems that canbe selected by the user. The look-up table can be a one-variable,two-variable or three-variable table; in each, the pH is calculated ormeasured based on the values of the other variables. A three-variabletable will have values for temperature, salt concentration (molar) andbase percent (%); a two-variable look-up table will have values for saltconcentration (molar) and base percent (%), and a one-variable look-uptable has values for base percent. The one-variable table can either bedirectly entered by the user or calculated by the software based on aninput value for the pK_(a) of the acid used. Typically, a two-variablelook-up table may have between 30-60 rows, and a one-variable look-uptable will have fewer rows. The two- and three-variable look-up tablesare constructed by manually preparing specific mixtures of acid and basebuffers, salt and water, and for the three-variable table, temperature.The pH of each mixture at the given conditions is measured and enteredinto the table.

The user also enters the molar concentrations for each of the acid, baseand salt stock solutions that will be combined at the pump in thesystem.

A final component of the user-entered information is the deliveredacid+base concentration. It has been found from experience that aconvenient ratio of acid+base percent and salt+solvent percent is about20% acid+base, and about 80% salt+solvent. However, these numbers arenot required, and can be adjusted according to the needs of the user andthe analytes being separated.

At a point in time, the pump in the chromatography systems is deliveringthe specified total flow rate of liquid to the separation column. Thesoftware calculates the pH and salt concentration at a particular timet, using the information provided in the gradient table and the look-uptable, and a fraction multiplier calculated using the particular pHcurve and/or salt concentration curve in the gradient table. Then, forthis particular pH and salt concentration, the % base, % acid, % saltand % solvent can be calculated, using interpolation, from valuesprovided in the pH look-up table. These values are sent to thechromatographic system to generate the specific mobile phase conditionsfor time t.

The method of the invention is further illustrated by way of thefollowing example. Note that an organic solvent can be substituted for asalt in the following calculations, for systems in which organicsolvents rather than salts are used.

TABLE 2 pH- Salt Gradient Table (provided by the user) time flow pH saltconc. salt conc. (min) (ml/min) pH curve (mM) curve T₀ = 0 1.0 (f₀) 5(pH₀) — 500 (sC₀) — T₁ = 10 1.0 (f₁) 6 (pH₁) 6 (pHC₁) 1000 (sC₁)  6(sCC₁) T₂ = 15 1.0 (f₂) 5 (pH₂) 6 (pHC₂) 500 (sC₀) 6 (sCC₂) T₃ = 16   0(f₃) 5 11 500 (sC₀) 11

TABLE 3.1 One Variable - pH Look-Up Table (empirical calibration table)base % pH  0 4.0  5 (bP₀) 5.0 (pHL₀) 10 (bP₁) 5.5 (pHL₁) 20 6.0

TABLE 3.2 Two Variables - pH Look-Up Table (empirical calibration table)Salt % Base % pH 20 (sPL₀)  5 (bP₀) 5.0 (pHL₀) 20 (sPL₀) 10 (bP₁) 5.5(pHL₁) 40 (sPL₁)  5 (bP₀) 5.2 (pHL₂) 40 (sPL₁) 10 (bP₁) 5.7 (pHL₃) . . .. . . . . .

TABLE 3.3 Three Variables - pH Look-Up Table (empirical calibrationtable) Temp. ° C. Salt % Base % pH 20° C. (Temp₀) 20 (sPL₀)  5 (bP₀) 5.0 (pHL₀) 20° C. (Temp₀) 20 (sPL₀) 10 (bP₁)  5.5 (pHL₁) 20° C. (Temp₀)40 (sPL₁)  5 (bP₀)  5.2 (pHL₂) 20° C. (Temp₀) 40 (sPL₁) 10 (bP₁)  5.7(pHL₃) 30° C. (Temp₁) 20 (sPL₀)  5 (bP₀) 5.05 (pHL₄) 30° C. (Temp₁) 20(sPL₀) 10 (bP₁) 5.56 (pHL₅) 30° C. (Temp₁) 40 (sPL₁)  5 (bP₀) 5.24(pHL₆) 30° C. (Temp₁) 40 (sPL₁) 10 (bP₁) 5.77 (pHL₇)

Molar Concentrations (Provided by the User):

acid concentration: acU=100 mMbase concentration: bcU=100 mMsalt concentration: scU=1000 mMacid and base concentration (bufferConcentrationU)=20 mM(bufferConcentrationU≦mM (acU, bcU)

Gradient Function

The gradient function is defined by the following set of equations:

y=y _(s)+(y _(n) −y ₅)×fraction  equation 1

where y is pH and salt concentration (or solvent, if an organic solventis used), and “fraction” is defined as follows (where values for t₁ andt₀ are obtained from the pH-salt gradient table, and t is the time ofinterest,

t ₀ ≦t≦t ₁):

$\begin{matrix}{\mspace{79mu} {{{{Curve}\mspace{14mu} 1\text{:}\mspace{14mu} {fraction}} = 1.0}\mspace{79mu} {{{Curve}\mspace{14mu} 2\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{8}}}\mspace{79mu} {{{Curve}\mspace{14mu} 3\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{5}}}\mspace{79mu} {{{Curve}\mspace{14mu} 4\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{3}}}\mspace{79mu} {{{Curve}\mspace{14mu} 5\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{2}}}\mspace{79mu} {{{Curve}\mspace{14mu} 6\text{:}\mspace{14mu} {fraction}} = \frac{t}{t_{1} - t_{0}}}\mspace{79mu} {{{Curve}\mspace{14mu} 7\text{:}\mspace{14mu} {fraction}} = \left( \frac{t}{t_{1} - t_{0}} \right)^{2}}\mspace{79mu} {{{Curve}\mspace{14mu} 8\text{:}\mspace{14mu} {fraction}} = \left( \frac{t}{t_{1} - t_{0}} \right)^{3}}\mspace{79mu} {{{Curve}\mspace{14mu} 9\text{:}\mspace{14mu} {fraction}} = \left( \frac{t}{t_{1} - t_{0}} \right)^{5}}\mspace{79mu} {{{Curve}\mspace{14mu} 10\text{:}\mspace{14mu} {fraction}} = \left( \frac{t}{t_{1} - t_{0}} \right)^{8}}{{{Curve}\mspace{14mu} 11\text{:}\mspace{14mu} {fraction}} = {{{0\mspace{14mu} {if}\mspace{14mu} t} < {\left( {t_{1} - t_{0}} \right)\mspace{14mu} {and}\mspace{14mu} {fraction}}} = {{1\mspace{14mu} {if}\mspace{14mu} t} = {\left( {t_{1} - t_{0}} \right).}}}}}} & {{equation}\mspace{14mu} 2}\end{matrix}$

pH and Salt (or Solvent) Concentration at any Gradient Time t

At any gradient time t, the pH value can be calculated from the gradientfunction, Equation 1 above, as follows:

From Table 2, the pH-Salt Gradient Table, y=pH, y_(s)=pH₀, andy_(n)=pH₁, for all values of t where t₀≦t≦t₁.

Using curve 6 as an example, the fraction is

$\frac{t}{t_{1} - t_{0}}.$

$\begin{matrix}{{{At}\mspace{14mu} {time}\mspace{14mu} t},{{{pH}(t)} = {{pH}_{0} + {\left( {{pH}_{1} - {pH}_{0}} \right) \times {\frac{t}{t_{1} - t_{0}}.}}}}} & {{equation}\mspace{14mu} 3}\end{matrix}$

Similarly, at time t, and using curve 6 as an example, the saltconcentration is calculated as:

$\begin{matrix}{{{sC}(t)} = {{sC}_{0} + {\left( {{sC}_{1} - {sC}_{0}} \right) \times {\frac{t}{t_{1} - t_{0}}.}}}} & {{equation}\mspace{14mu} 4}\end{matrix}$

Calculation of Base Percent

1) Using the one-variable pH Look-up table (Table 3.1), the base percentcan be calculated, using interpolation, as follows:

$\begin{matrix}{{BasePercent} = {{bP}_{0} + {\frac{{bP}_{1} - {bP}_{0}}{{{pH}\; L_{1}} - {{pH}\; L_{0}}} \times \left( {{pH} - {{pH}\; L_{0}}} \right)}}} & {{equation}\mspace{14mu} 5.1}\end{matrix}$

2) For a given salt percent (sP) and pH, where

sPL ₀<sP<sPL ₁, pHL ₀<pH<pHL ₁, and pHL ₂<pH<pHL ₃,

the base percent (%) can be calculated from the values provided by theuser in the two-variable pH Look-up table (Table 3.2), usinginterpolation:

$\begin{matrix}{\mspace{79mu} {{{bPI}_{0} = {{bP}_{0} + {\frac{{bP}_{1} - {bP}_{0}}{{{pH}\; L_{1}} - {{pH}\; L_{0}}} \times \left( {{pH} - {{pH}\; L_{0}}} \right)}}}\mspace{79mu} {{bPI}_{1} = {{bP}_{0} + {\frac{{bP}_{1} - {bP}_{0}}{{{pH}\; L_{3}} - {{pH}\; L_{2}}} \times \left( {{pH} - {{pH}\; L_{2}}} \right)}}}{{{then}\mspace{14mu} {basePercent}} = {{bPI}_{0} + {\frac{{bPI}_{1} - {bPI}_{0}}{{sPL}_{1} - {sPL}_{0}} \times \left( {{sP} - {sPL}_{0}} \right)}}}}} & {{equation}\mspace{14mu} 5.2}\end{matrix}$

3) For a given temperature (Temp), salt percent (sP) and pH, where

Temp₀<Temp<Temp₁,

sPL ₀<sP<sPL ₁,

pHL ₀<pH<pHL ₁,

pHL ₂<pH<pHL ₃,

pHL ₄<pH<pHL ₅,

pHL ₆<pH<pHL ₇,

the base percent (%) can be calculated from the values provided by theuser in the three-variable pH Look-up table (Table 3.3), usinginterpolation:

$\begin{matrix}{\mspace{79mu} {{{bPI}_{0} = {{bP}_{0} + {\frac{{bP}_{1} - {bP}_{0}}{{pHL}_{1} - {pHL}_{0}} \times \left( {{pH} - {{pH}\; L_{0}}} \right)}}}\mspace{79mu} {{bPI}_{1} = {{bP}_{0} + {\frac{{bP}_{1} - {bP}_{0}}{{pHL}_{3} - {pHL}_{2}} \times \left( {{pH} - {pHL}_{2}} \right)}}}\mspace{79mu} {{bPI}_{2} = {{bP}_{0} + {\frac{{bP}_{1} - {bP}_{0}}{{pHL}_{5} - {pHL}_{4}} \times \left( {{pH} - {pHL}_{4}} \right)}}}\mspace{79mu} {{bPI}_{3} = {{bP}_{0} + {\frac{{bP}_{1} - {bP}_{0}}{{pHL}_{7} - {pHL}_{6}} \times \left( {{pH} - {pHL}_{6}} \right)}}}\mspace{79mu} {{bPI}_{4} = {{bPI}_{0} + {\frac{{bPI}_{1} - {bPI}_{0}}{{sPL}_{1} - {sPL}_{0}} \times \left( {{sP} - {sPL}_{0}} \right)}}}\mspace{79mu} {{bPI}_{5} = {{bPI}_{2} + {\frac{{bPI}_{3} - {bPI}_{2}}{{sPL}_{1} - {sPL}_{0}} \times \left( {{sP} - {sPL}_{0}} \right)}}}\mspace{79mu} {and}{{basePercent} = {{bPI}_{4} + {\frac{{bPI}_{5} - {bPI}_{4}}{{Temp}_{1} - {Temp}_{0}} \times \left( {{Temp} - {Temp}_{0}} \right)}}}}} & {{equation}\mspace{14mu} 5.3}\end{matrix}$

Calculation of % A, % B, % C and % D in the Total Mixture (Sent toGradient Proportioning Valve by Software) 1) Calculation of Salt PercentMax

acidPercent×aCU+basePercent×bCU=100%×bufferConcentrationU  eq. 5.4.1

acidPercent+basePercent+saltPercent+aqueousPercent=100%  eq. 5.4.2

From equations 5.4.1 and 5.4.2 the following can be derived:

$\begin{matrix}{\mspace{79mu} {{acidPercentMax} = {100\% \times \frac{bufferConcentrationU}{aCU}}}} & {{eq}{.5}{.4}{.3}} \\{\mspace{79mu} {{{basePercentMax} = {100\% \times \frac{bufferConcentrationU}{bCU}}}\mspace{79mu} {and}}} & {{eq}{.5}{.44}} \\{{saltPercentMax} = {{100\%} - \left( {\max \mspace{14mu} {of}\mspace{14mu} {acidPercentMax}\mspace{14mu} {and}\mspace{14mu} {basePercentMax}} \right)}} & {{eq}{.5}{.45}}\end{matrix}$

2) Calculation of Solvent Percentages Sent to Pump:

From equations 5.1, 5.2 or 5.3 basePercent is obtained. From this value,the following can be calculated:

$\begin{matrix}{{{acidPercent} = \frac{{100\% \times {bufferConcentrationU}} - {{basePercent} \times {bCU}}}{aCU}}\mspace{79mu} {{{From}\mspace{14mu} {Equation}\mspace{14mu} 4},{{saltPercent}\mspace{14mu} {is}\mspace{14mu} {calculated}\mspace{14mu} {as}\text{:}}}} & {{eq}{.6}{.1}} \\{\mspace{79mu} {{{saltPercent} = {\frac{sC}{sCU} \times 100\%}}{{{if}\mspace{14mu} \left( {{saltPercent} > {saltPercentMax}} \right)\mspace{14mu} {then}\mspace{14mu} {saltPercent}} = {saltPercentMax}}}} & {{eq}{.6}{.2}} \\{{aqueousPercent} = {{100\%} - {acidPercent} - {basePercent} - {saltPercent}}} & {{eq}{.6}{.3}}\end{matrix}$

The values for acidPercent, basePercent, saltPercent and aqueousPercentcan be assigned to solvent A, B, C and D in the pumps.

Calculations for a one-variable look-up table from pK_(a)

For a given pK_(a) value

pH_(min)=pK_(a)−1

pH_(max)=pK_(a)+1

numPH=40

for arrayIndex=0 to arrayIndex=39,

$\begin{matrix}{{{pH}\lbrack{arrayIndex}\rbrack} = {{pH}_{\min} + {\left( {{pH}_{\max} - {pH}_{\min}} \right) \times \frac{arrayIndex}{{numPH} - 1}}}} & {{equation}\mspace{14mu} 8} \\{{{basePercent}\lbrack{arrayIndex}\rbrack} = {\frac{10^{- {pK}_{a}}}{10^{- {pK}_{a}} + 10^{- {{pH}{\lbrack{arrayIndex}\rbrack}}}} \times 100\%}} & {{equation}\mspace{14mu} 9}\end{matrix}$

So, for example, for a pK_(a)=6.8, pH_(min)=5.8, pH_(max)=6.5 andnumPH=40,

a one-variable pH look-up table can be obtained from equations 8 and 9:

TABLE 4 one-variable pH look-up Base % pH 9.09% 5.8 . . . . . .  50% 6.8. . . . . . 90.9% 7.8

The acid and base components can theoretically be any acid and base, butpreferably a weak acid is paired with its conjugate base, or a weak baseis paired with its conjugate acid. An example of an acid-base pair isTRIS and TRIS chloride. Other acid-base pair are also suitable and arewell known to those skilled in the art.

The salt component preferably is any salt which is neutral to the liquidsystem, i.e. does not react in the system by any other way than by ionicdissociation. Preferable examples are NaCl or KCl. However, theinvention could easily be extended to the use of pH active salts, e.g.ammonium sulphate, and it is considered to be within the knowledge ofthe person skilled in the art to make the corresponding modifications tothe entered variables.

Combinations of acids, combinations of bases, and combinations of saltscan also be used.

The solvent component can be any solvent or combination of solvents inwhich the other components of the liquid mixture are soluble. In oneembodiment, the solvent is preferably distilled and/or deionized water.In another embodiment, for example in reverse phase chromatographyseparations, a water and acetonitrile mixture is preferred.

In other systems, other organic solvents may be used. Typically, when asolvent other than water is used, it preferably is in admixture withwater, whereby the pH of the solvent may be taken to be that of thewater phase.

In a preferred embodiment, e.g. in a liquid chromatographic system, thecalculations are carried out by a data program, implemented from theequations given herein above, governing directly a metering device, suchas a pump and valve system, or any other equivalent means of deliveringthe components to the chromatography device. The program preferablyincludes the ability to correct for separations carried out at differenttemperatures, by including temperature in the empirically-derivedlook-up table (as in Table 3.3). As will be understood by one skilled inthe art, the invention is not limited to any particular flow rate, anyparticular mixing system, or any particular method of generating a flowthrough a chromatographic device. For example, the method of theinvention can be used in both low pressure liquid chromatography (wheremixing occurs prior to pumping), and high pressure liquidchromatography, where pumping occurs prior to mixing.

Example Ion Exchange Separation of Monoclonal Antibody

A commercial preparation of a chimeric monoclonal antibody was analyzedwith cation exchange chromatography. The described invention was used toprovide different separation buffers to adjust resolution in theseparation.

As shown in FIG. 2, the instrument method specified that separationshould occur at pH 6.0 with a linear gradient from 0 to 0.5M sodiumchloride. Using a specified pK of 6.8, the software calculated that theinstrument should blend the mobile phase as 17.26% A and 2.74% B whilechanging over time from 80% C:0% D to 30% C:50% D.

To improve this separation, a second instrument method was createdspecifying that the same gradient be used at a pH of 6.8. Using the pKof 6.8, the software calculated that the instrument should blend themobile phase as 10.0% A and 10.0% B while changing over time from 80%C:0% D to 30% C:50% D. The resulting separation is shown in FIG. 3.Retention is lower, and more separated species are apparent.

Experimental Conditions:

Sample: Chimeric Monoclonal Antibody, 4 mg/mL

Injection: 10 uL Column: Waters Protein-Pak Hi Res SP, 7 mm, 4.6×100 mm

Instrument: Waters ACQUITY UPLC H-Class System with TUV detector at 280nm

Buffers: A: 0.125M NaH₂PO₄

-   -   B: 0.125M Na₂HPO₄    -   C: 1.0 M NaCl    -   D: Water

Gradient Table 1 Time Flow pH Salt Salt (min) (mL/min) pH Curve Conc.Curve Init. 1.0 6.0 . . . 0.0 . . . 2 1.0 6.0 6 0.0 6 32 1.0 6.0 6 0.5 632.1 1.0 6.0 6 0.0 6 47 1.0 6.0 6 0.0 6

Gradient Table 2 Time Flow pH Salt Salt (min) (mL/min) pH Curve Conc.Curve Init. 1.0 6.8 . . . 0.0 . . . 2 1.0 6.8 6 0.0 6 32 1.0 6.8 6 0.5 632.1 1.0 6.8 6 0.0 6 47 1.0 6.8 1 0.0 6

What is claimed is:
 1. A method of preparing a liquid mixture, themixture comprising one or more acids, one or more bases, one or moresalts, and one or more solvents, the method comprising the steps of: i)calculating pH and/or salt concentration at a particular time t from auser-determined gradient function; and ii) based on the values obtainedin step (i), calculating percent acid, percent base, percent salt andpercent solvent in the liquid mixture at time t.
 2. The method of claim1, wherein the gradient function comprises a fraction multiplier thatprovides predetermined changes in pH and/or salt concentration, eachbeing independent of the other, over a time period of interest.
 3. Themethod according to claim 1 or claim 2, wherein the gradient function isbased on the following equation:y=y _(s)+(y _(n) −y _(s))×fraction  (equation 1) where y is selectedfrom the group consisting of pH and salt concentration, y_(s) is thevalue at time 1 and y_(n) is the value at time
 2. 4. The methodaccording to any one of claims 1-3, wherein the fraction multiplier isselected from the group consisting of:$\mspace{79mu} {{{1\text{:}\mspace{14mu} {fraction}} = 1.0},\mspace{79mu} {{2\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{8}}},\mspace{79mu} {{3\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{5}}},\mspace{79mu} {{4\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{3}}},\mspace{79mu} {{5\text{:}\mspace{14mu} {fraction}} = {1 - \left( {1 - \frac{t}{t_{1} - t_{0}}} \right)^{2}}},\mspace{79mu} {{6\text{:}\mspace{14mu} {fraction}} = \frac{t}{t_{1} - t_{0}}},\mspace{79mu} {{7\text{:}\mspace{14mu} {fraction}} = \left( \frac{t}{t_{1} - t_{0}} \right)^{2}},\mspace{79mu} {{8\text{:}\mspace{14mu} {fraction}} = \left( \frac{t}{t_{1} - t_{0}} \right)^{3}},\mspace{79mu} {{9\text{:}\mspace{14mu} {fraction}} = \left( \frac{t}{t_{1} - t_{0}} \right)^{5}}}$$\mspace{79mu} {{10\text{:}\mspace{14mu} {fraction}} = {\left( \frac{t}{t_{1} - t_{0}} \right)^{8}\mspace{14mu} {and}}}$11:  fraction = 0  if  t < (t₁ − t₀)  and  fraction = 1  if  t = (t − 1 − t₀).5. The method according to any one of claims 1-4, wherein for aparticular pH or salt concentration the % base can be calculated, usinginterpolation, from values provided in a pH look-up table.
 6. The methodaccording to claim 5, wherein the pH look-up table is empiricallyderived or is calculated from the pK_(a) of the acid.
 7. The methodaccording to claim 6, wherein the pH look-up table provides pH valuesfor a given acid+base percentage, ratio, or relative concentration. 8.The method according to claim 6, wherein the pH look-up table providespH values for a given acid+base percentage, ratio, or relativeconcentration, and salt concentration.
 9. The method according to claim6, wherein the pH look-up table provides pH values for a given acid+basepercentage, ratio, or relative concentration, salt concentration andtemperature.
 10. The method according to any one of claims 1-4, whereinthe proportions of acid, base, salt and solvent are used for controllinga liquid mixture device.
 11. The method according to any one of claims1-5, wherein liquid mixtures are prepared from stock solutions of atleast one acid, at least one base, at least one salt, and at least onesolvent.
 12. A computer program for determining relative proportions ina liquid mixture according to any one of the preceding claims, whereinthe proportions are used for controlling a liquid mixture preparationdevice.
 13. An eluent preparation device comprising i) a liquid mixturepreparation device comprising a mixed liquid outlet port and a pluralityof inlet ports connected to component sources of at least one of anacid, a base, a salt, and a solvent, and ii) a mixer control unitarranged to control the relative component proportions supplied throughthe inlet ports of the liquid mixture preparation device, wherein themixer control unit i) calculates pH and salt concentration from agradient function; and ii) based on the values obtained in step (i),calculates percent acid, percent base, percent salt and percent solventin the liquid mixture.
 14. A liquid chromatography system comprising theeluent preparation device of claim
 13. 15. The liquid chromatographysystem of claim 9 wherein the system comprises a low-pressure mixingsystem.
 16. The liquid chromatography system of claim 9 wherein thesystem comprises a high-pressure mixing system.